Targeted expansion of Qa-1-peptide-specific regulatory CD8 T cells to ameliorate arthritis

ABSTRACT

Nanoparticles to treat autoimmune diseases and HIV infection are provided. The nanoparticles comprise a biocompatible polymer and a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to a peptide, and wherein the HLA-E-peptide complex is linked to the surface of the nanoparticle. The present invention also relates to methods for treating autoimmune diseases and HIV infection.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application PCT/US2013/061851, filed Sep. 26, 2013, whichwas published under PCT Article 21(2) in English, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos.61/707,357, filed Sep. 28, 2012, and 61/773,959, filed Mar. 7, 2013, thecontent of each referenced application is incorporated by referenceherein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersCA070083 and AI037562 awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to nanoparticles and methods for treatingautoimmune diseases.

BACKGROUND OF THE INVENTION

Achieving a balance between induction of protective immunity againstpathogens and maintenance of self-tolerance is a central feature of theadaptive immune system. Although negative selection in the thymusremoves the majority of clones that express T cell receptors (TCR) withhigh affinity for self-peptide MHC products, this process is incomplete.A significant fraction of mature peripheral T cells that respond toself-peptide-MHC complexes may differentiate into effector cells in thecontext of inflammatory stimuli (Bouneaud et al., 2000; Goldrath andBevan, 1999; Slifka et al., 2003). Although this process is constrainedby abortive or defective TCR signaling resulting in cellular elimination(AICD) or inactivation (Martin et al., 1999; Kearney et al., 1994),these cell-intrinsic mechanisms may not suffice to prevent thedevelopment of autoimmune disorders (Anderton et al., 2001;Panoutsakopoulou et al., 2001). There is increasing evidence thatself-tolerance may also depend on inhibitory interactions betweeneffector T cells and regulatory or suppressive cells (Littman andRudensky, 2010). A regulatory subset of CD8+ T cells, termed CD8+ Tregcells, has been found to inhibit follicular T helper cell responses,which are essential for production of autoantibodies and formation ofectopic germinal centers (GC). However, the potential contribution ofCD8+ Treg cells to the pathogenesis and treatment of autoimmune diseaseis not well understood. Due to the severity and breath of autoimmunediseases such as lupus and rheumatoid arthritis, there is a great needfor effective treatments of such diseases.

SUMMARY OF THE INVENTION

The present invention, in one aspect, relates to the development ofstrategies based on in vivo and in vitro expansion and activation of theCD8+ Treg cells for the treatment of autoimmune diseases.

In some aspects provided, is a composition for a nanoparticle comprisinga biocompatible polymer and a complex, wherein the complex is a majorhistocompatibility complex (MHC) class I antigen E (HLA-E) linked to apeptide, and wherein the HLA-E-peptide complex is linked to the surfaceof the nanoparticle. In certain embodiments, at least 4 units, at least5, at least 6, at least 7, at least 8, at least 9 or at least 10 unitsof the HLA-E-peptide complex are linked together to form a moiety andthe moiety is linked to the surface of the nanoparticle. In certainembodiments of the invention, the HLA-E-peptide complex of thisnanoparticle composition comprises biotin, and the complexes are linkedtogether through biotin-avidin interaction to form the moiety. Incertain embodiments of the invention, the peptide linked to the HLA-E isselected from the group consisting of Hsp60_(p216), B7sp and FL9. Incertain embodiments, the HLA-E-peptide complex or the moiety is linkedto the surface of the nanoparticle via PEGylation.

In certain embodiments, the peptide of this complex is linked to HLA-Evia a flexible linker. In certain embodiments, wherein the peptide islinked to HLA-E via a flexible linker, this linker is a Gly-Ser linker.

In certain embodiments, the heavy chain and light chain (β-2microglobulin) of the HLA-E are linked via a flexible linker. In certainembodiments, wherein the heavy chain and light chain (β-2 microglobulin)of the HLA-E are linked via a flexible linker, the flexible linker is aGly-Ser linker.

In certain embodiments, the biocompatible polymer of the nanoparticle isselected from the group consisting of poly(lactic-co-glycolic acid)(PLGA), poly(ethylene glycol) (PEG), chitosan, and chitosan-PEG. Incertain embodiments of the nanoparticle composition, a low releasingdose of IL-15 is incorporated into the nanoparticle.

In other aspects provided, is a method for treating an autoimmunedisease comprising: administering to a subject in need of such treatmentnanoparticles, as described in the embodiments above, in an amounteffective to ameliorate a symptom of the autoimmune disease. In certainembodiments of the invention, the autoimmune disease treated by theadministration of nanoparticles is selected from a group consisting ofsystemic lupus erythematosus, chronic graft versus host disease,rheumatoid arthritis, insulin-dependent diabetes mellitus, multiplesclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome,Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia,hyperviscosity syndrome, monoclonal gammopathy of undetermined origin,POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease.

In other aspects provided, is a method for treating an autoimmunedisease comprising: contacting dendritic cells isolated from a subjectin need of such treatment with Hsp60_(p216) peptide to generateHsp60_(p216)-loaded dendritic cells; and administering to the subjectthe Hsp60_(p216)-loaded dendritic cells in an amount effective toameliorate a symptom of the autoimmune disease. In certain embodimentsthe dendritic cells isolated from a subject in need of such treatmentare contacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16, 18 or 24 hoursto generate Hsp60_(p216)-loaded dendritic cells. In certain embodimentsof the invention, this method is used to treat autoimmune diseasesselected from the group consisting of systemic lupus erythematosus,chronic graft versus host disease, rheumatoid arthritis,insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis,inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn'sdisease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome,monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma,macroglobulinemia, and cold agglutinin disease.

In other aspects provided, is method for treating an autoimmune diseasecomprising: isolating from a subject in need of such treatment CD8+ Tcells that bind Hsp60_(p216) peptide; growing the isolated cells in aculture medium containing IL-15C until the number of CD8+ T cells thatbind Hsp60_(p216) peptide increases to at least 3-5% of CD8+ T cells,thereby producing a population of cells enriched with CD8+ T cells thatbind Hsp60_(p216) peptide; and administering CD8+ T cells that bindHsp60_(p216) peptide from the described population of cells to thesubject in an amount effective to ameliorate a symptom of the autoimmunedisease. In certain embodiments, the step of isolating CD8+ T cells thatbind Hsp60_(p216) peptide comprises sorting a sample containing T cellsobtained from the subject into CD8+ T cells that bind Hsp60_(p216)peptide using a fluorescently labeled moiety having at least 4 units ofa complex, wherein the complex is a major histocompatibility complex(MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide. Incertain embodiments, the HLA-E-peptide complex described comprisesbiotin, and at least 4 such complexes associate through biotin-avidininteraction to form the moiety. In certain embodiments, avidin isfluorescently labeled. In certain embodiments, the fluorescent labelused to label avidin is Phycoerythrin (PE). In certain embodiments, thepopulation of cells enriched with CD8+ T cells that bind Hsp60_(p216)peptide is further enriched using anti-R-Phycoerythrin (PE) microbeads.In certain embodiments, the autoimmune disease treated through thesesteps is selected from the group consisting of systemic lupuserythematosus, chronic graft versus host disease, rheumatoid arthritis,insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis,inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn'sdisease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome,monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma,macroglobulinemia, and cold agglutinin disease.

In some aspects provided, is a method for treating HIV infection. Themethod comprises administering to a subject in need of such treatmentnanoparticles described herein in an amount effective to ameliorate asymptom of the HIV infection.

In some aspects provided, is a method for treating HIV infection. Themethod comprises contacting dendritic cells isolated from a subject inneed of such treatment with Hsp60_(p216) peptide to generateHsp60_(p216)-loaded dendritic cells; and administering to the subjectthe Hsp60_(p216)-loaded dendritic cells in an amount effective toameliorate a symptom of the HIV infection. In certain embodiments, thedendritic cells are contacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16,18 or 24 hours to generate Hsp60_(p216)-loaded dendritic cells.

In some aspects provided, is a method for treating HIV infectioncomprising: isolating from a subject in need of such treatment CD8+ Tcells that bind Hsp60_(p216) peptide; growing the isolated cells in aculture medium containing IL-15C until the number of CD8+ T cells thatbind Hsp60_(p216) peptide increases to at least 3-5% of CD8+ T cells,thereby producing a population of cells enriched with CD8+ T cells thatbind Hsp60_(p216) peptide; and administering CD8+ T cells that bindHsp60_(p216) peptide from the population of cells to the subject in anamount effective to ameliorate a symptom of the HIV infection. Incertain embodiments, the step of isolating CD8+ T cells that bindHsp60_(p216) peptide comprises sorting a sample containing T cellsobtained from the subject into CD8+ T cells that bind Hsp60_(p216)peptide using a fluorescently labeled moiety having at least 4 units ofa complex, wherein the complex is a major histocompatibility complex(MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide. Incertain embodiments, the HLA-E-peptide complex described comprisesbiotin, and at least 4 such complexes associate through biotin-avidininteraction to form the moiety. In certain embodiments, avidin isfluorescently labeled. In certain embodiments, the fluorescent labelused to label avidin is Phycoerythrin (PE). In certain embodiments, thepopulation of cells enriched with CD8+ T cells that bind Hsp60_(p216)peptide is further enriched using anti-R-Phycoerythrin (PE) microbeads.

Each of the embodiments and aspects of the invention can be practicedindependently or combined. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the invention can encompass variousembodiments as will be understood.

All documents identified in this application are incorporated in theirentirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is graph showing that selective expansion of Qa-1-restrictedCD8+ Treg inhibits arthritis. Arthritis was induced in C57BL/6 mice asdescribed in Methods. Irradiated LPS-activated Kb^(−/−)Db^(−/−) bonemarrow-derived DC pre-loaded with or without indicated peptides wasinjected subcutaneously at the tail base at d27, d36 and d49 ( ).Arthritis scores are shown; 5-6 mice per group. Group (DC alone) versusgroup (Hsp60p216-DC), *P<0.05.

FIG. 1B is a plots showing the selective expansion of Qa-1-restrictedCD8 Treg, where CII-immune B6 mice were immunized withKb^(−/−)Db^(−/−)DC loaded with or without the indicated peptide asdescribed in FIG. 1A. Qdm- or Hsp60p216-CD8 cells from draining lymphnodes were analyzed at d42 by staining with RA.Qa-1-Qdm-tetramer orWT.Qa-1-Hsp60p216-tetramer, respectively. Representative FACS plots areshown.

FIG. 2A is a plot showing that selective expansion of Qa-1-restrictedCD8+ Treg inhibited arthritis. Hsp60p216-tet+ and Hsp60p216-tet− CD8cells were sorted from CII-immune B6 mice before incubation in IL-15C(10 ng ml-1)*10d. These cells were then incubated with Hsp60p216tetramer and enriched by anti-PE microbeads. FACS analysis of tetramerstaining of these cells indicated that incubation of CD8 cells that weretet+ but not tet− resulted in a substantial number of Hsp60p216 tet+cells.

FIG. 2B is a graph showing the transfer of Qa-1-Hsp60_(p216) tetramer⁺CD8 T cells. The Hsp60p216-tet+ or Hsp60p216-tet− fraction of CD8 cellswas transferred into Rag2^(−/−)Prf1^(−/−) mice along with CD4 and Bcells from arthritic mice. Arthritis was induced as described inMethods. Arthritis scores are shown; 3 mice per group.

FIG. 2C is a bar graph showing anti-mouse CII IgG titers at d30; 3 miceper group. Group (tet− CD8) versus group (tet+ CD8), *P<0.05.

FIG. 2D is a bar graph showing anti-mouse CII IgG titers at d30. HereQdm-tet+ or Qdm-tet− CD8 cells were transferred intoRag2^(−/−)Prf1^(−/−) mice along with CD4 and B cells from arthriticmice.

FIG. 2E is a bar graph showing selective expansion of Qa-1-restrictedCD8 Treg. Hsp60p216-tet+ CD8 cells were enumerated at d34 after adoptivetransfer as described in FIG. 2B.

FIG. 3 shows the amino acid sequence of human IL-15 (SEQ ID NO: 3; NCBIReference Sequence: NP_000576.1).

DETAILED DESCRIPTION OF THE INVENTION

A subpopulation of CD8+ regulatory T cells is essential for maintenanceof self-tolerance and prevention of autoimmune disease (WO 2012/054509incorporated by reference herein). These CD8+ Treg cells are programmedto suppress rather than activate immunity and represent an essentialregulatory element of the immune response and a guarantor ofself-tolerance. The present invention, in one aspect, relates to thedevelopment of strategies based on in vivo and in vitro expansion andactivation of the CD8+ Treg cells for the treatment of autoimmunediseases.

The specialized regulatory CD8+ T cells selectively suppress CD4+follicular helper T cell (TFH) activity through perforin-dependent lysisby recognition of class Ib major histocompatibility complex (MHC)peptide Qa-1 (mouse homolog of human leukocyte antigen E (HLA-E))expressed at the surface of TFH cells and dampen autoantibody responses.Qa-1, the mouse homolog of human leukocyte antigen E (HLA-E), forms aheterodimer with β₂-microglobulin that binds to and presents peptidesderived from self or foreign proteins after deliberate immunization orinfection (Lo et al., 1999; Lo et al., 2000; Sullivan et al., 2002).

Three peptides are presented by Qa-1 (HLA-E): Qdm (B7sp in humans),Hsp60_(p216) and FL9. Qa-1 heterodimers containing peptides derived fromMHC class Ia leader sequences, called Qdm (for Qa-1 determinantmodifier), bind to nonclonally distributed CD94-NKG2A receptorsexpressed by natural killer (NK) cells and a subpopulation of CD8+ Tcells. The functional consequence of Qa-1/Qdm-NKG2A interactions isgenerally inhibition of NK or CD8 cytolytic activity (Moser et al.,2002). Hsp60_(p216) peptide (GMKFDRGYI; SEQ ID NO: 1) contains residues216-224 of the 60 kD heat shock protein (Hsp). The Hsp60_(p216) peptidecan efficiently replace Qdm peptides under conditions of cellularactivation and stress. A third peptide that is presented by Qa-1 underconditions of defective antigen processing in the endoplasmic reticulum(ERAAP dysfunction) is the highly conserved peptide FL9, FYAEATPML (SEQID NO: 2), which is derived from the conservative Fam49b gene.Recognition of Qa-1/FL9 by CD8 T cells may serve to eliminate abnormalcells that have defects in antigen presentation.

One of the potential platforms for specific expansion of Qa-1-restrictedor HLA-E-restricted CD8+ Treg is based on vaccination or treatment withpeptide-Qa-1(HLA-E)-coated nanoparticles (p-Qa-1(HLA-E)-NP).Administration of nanoparticles conjugated with Qa-1 tetramers withoutco-stimulatory activity is competent to stimulate Qa-1-peptide-specificCD8 T cells, since CD8+ Treg are memory cells according to function andphenotype. Unlike naïve CD8 cells, memory cells can be stimulated toexpand by antigen presented without co-stimulation.

Accordingly, aspects of the invention involve nanoparticles (NP)comprising a biocompatible polymer and a complex of MHC class I antigenE (HLA-E) linked to a peptide. The HLA-E-peptide complex is linked tothe surface of the nanoparticle to ensure efficient exposure to TCR onCD8 cells. To enhance the efficiency of p-Qa-1(HLA-E)-NP-mediatedbinding and expansion of CD8 Treg in vivo, in some embodiments, at least4 units, at least 5, at least 6, at least 7, at least 8, at least 9 orat least 10 units of the HLA-E-peptide complex are linked together toform a moiety and the moiety is then linked to the surface of thenanoparticle. In some embodiments, the HLA-E-peptide complex comprisesbiotin, and the complexes are linked together through biotin-avidininteraction to form the moiety. The moiety is conjugated to the carboxylgroups on the NP surface using carbodiimide coupling chemistry (EDC/NHS)to generate p-Qa-1 (HLA-E)-NP particles.

In some embodiments, the HLA-E-peptide complex or the moiety is linkedto the surface of the nanoparticle via PEGylation. PEGylation of NPhelps prevent internalization of p-Qa-1-NP by phagocytes. Covalentattachment of p-Qa-1(HLA-E) to the distal end of individual PEGmolecules rather than to the NP core enables efficient exposure to TCRon CD8+ cells. Alternatively, low molecular-weight chitosan (LMWC) canbe adopted as an alternative surface coating method. LMWC also provideshydrophilic layers on NP surface similar to PEG, reducing opsonizationand phagocytic uptake of PLGA-LMWC by macrophages (Amoozgar Z. Mol Pharm2012, Mochizuki M. FASEB 2003).

The NP can be synthesized using any biocompatible polymer, including butnot limited to, poly(lactic-co-glycolic acid) (PLGA), poly(ethyleneglycol) (PEG), chitosan, and chitosan-PEG. In some embodiments, PLGA isemployed to synthesize the NP for the delivery of p-Qa-1(HLA-E). PLGA isan FDA approved drug delivery reagent and displays favorable featuresamong other available NP particles (Fe304-PEG, Quantum dots, orLiposomes), including controllable biodegradability, excellentbiocompatibility, and high safety. PLGA-b-PEG copolymers can besynthesized by direct conjugation of PLGA-COOH with NH2-PEG-COOH, asdescribed by Cheng et al. (Formulation of functionalized PLGA-PEGnanoparticles for in vivo targeted drug delivery. Biomaterials 28,869-876 (2007)). The carboxyl group in the copolymer is located at theterminal end of the hydrophilic group allowing availability for surfacechemistry.

In some embodiments, the peptide is linked to the HLA-E via a flexiblelinker. In some embodiments, the heavy chain and light chain (β-2microglobulin) of the HLA-E are linked via a flexible linker. It isunderstood and herein contemplated that any linker known by those ofskill in the art can be used to make the disclosed NP. The choice ofwhich linker to use can be determined by those of skill in the art basedon the desired length and flexibility of the linker. Thus, in someembodiments, the linker can be any short peptide sequence as long as itis hydrophilic and forms a flexible linker by not building a secondarystructure that interferes with the main domain structures. In someembodiments, glycine-serine, the most commonly used flexible linker forthe production of recombinant single chain protein composed of differentmoieties, is used. The Gly-Ser linker can be but is not limited to(Gly4Ser)n or (Gly3Ser)n. Thus, for example, the linker can be G4S,(G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (G4S)7, (G4S)8, (G4S)9, (G4S)10,G3S, (G3S)2, (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G3S)7, (G3S)8, (G3S)9,(G3S)10. It is further understood that the linkers used can be acombination of chemical and gly-ser linkers. For example, the linkerbetween the peptide and the HLA-E can be a chemical linker and thelinker between the heavy and light chains of the HLA-E can be a Gly-Serlinker. It is further understood that the for greater flexibility, alonger linker is used; whereas, a shorter linker is used for applicationwhere more rigidity is needed. Typically, the linker between the peptideand the HLA-E is 10-20 amino acids long, while the linker between theheavy and light chains of the HLA-E is 15-25 amino acids long.Specifically contemplated herein are NP wherein the linker between thepeptide and the β-2 microglobulin of the HLA-E is composed of 15 aminoacids [(G4S)3] and β2-microglobulin and the heavy chain of HLA-E isconnected by a linker composed of 20 amino acids [(G4S)4].

CD8+ Treg cells depend on IL-15 for activation of suppressive activity.Thus, in some embodiments, a low releasing dose of IL-15 is incorporatedinto the nanoparticle. The low releasing dose of IL-15 helps promote theexpansion of Qa-1(HLA-E)/peptide specific CD8 Treg cells uponstimulation by p-Qa-1(HLA-E). The amino acid sequence of human IL-15 isrepresented by NCBI Reference Sequence: NP_000576.1 (SEQ ID NO: 3).IL-15 polypeptides also include fragments of IL-15, such as amino acids49-162 of SEQ ID NO:3, which has previously been characterized as amature form of IL-15 derived by proteolytic cleavage of a leadersequence from the polypeptide of NP_000576.1, and other fragments thatretain the biological activity of IL-15 are encompassed by the termIL-15.

The nanoparticles described herein can be used to treat autoimmunediseases. Accordingly, aspects of the invention involve a method fortreating an autoimmune disease by administering to a subject in need ofsuch treatment nanoparticles described herein in an amount effective toameliorate a symptom of the autoimmune disease. In some embodiments, thepeptide in the nanoparticles is Hsp60_(p216) Other aspects of theinvention involve a method for treating an autoimmune disease byadministering to a subject in need of such treatment a complexcomprising a peptide conjugated to any biocompatible polymer, such as,but not limited to poly(lactic-co-glycolic acid) (PLGA), poly(ethyleneglycol) (PEG), chitosan, and chitosan-PEG. In some embodiments, thepeptide is Hsp60_(p216) In some embodiments, the complex comprisesHsp60_(p216) conjugated to PEG.

Expansion of CD8+ Treg cells specific for Qa-1(HLA-E)-Qdm orQa-1(HLA-E)-Hsp60_(p216) in vivo can also be induced by administeringdendritic cells pulsed with either peptide. It has been discovered thatimmunization with Hsp60_(p216)-loaded dendritic cells (DC) efficientlyinhibits the development of collagen-induced arthritis (CIA).Accordingly, aspects of the invention include a method for treating anautoimmune disease. The method comprises contacting dendritic cellsisolated from a subject in need of such treatment with Hsp60_(p216)peptide to generate Hsp60_(p216)-loaded dendritic cells; andadministering to the subject the Hsp60p216-loaded dendritic cells in anamount effective to ameliorate a symptom of the autoimmune disease.

The term “contacting dendritic cells isolated from a subject in need ofsuch treatment with Hsp60_(p216) peptide” includes any means ofcontacting including but not limited to, mixing the Hsp60_(p216) peptideand the cells in medium, mixing the Hsp60_(p216) peptide in liposomeswith the cells or expressing the Hsp60_(p216) peptide from a recombinantnucleic acid in the cells. In some embodiments, the dendritic cells arecontacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16, 18 or 24 hours togenerate Hsp60_(p216)-loaded dendritic cells.

The term “Hsp60_(p216)-loaded dendritic cell” as used herein refers to adendritic cell presenting Hsp60_(p216) on its surface in a mannereffective to selectively expand CD8+ Treg cells that specificallyrecognize Hsp60_(p216) The dendritic cell may become loaded with thepeptide by directly binding the peptide from the medium on its surfaceor by processing the peptide intra-cytoplasmically before presenting thepeptide. Processing the peptide may include proteolytically generatingthe presented peptide from a longer peptide.

Some aspects of the invention involve a method for treating anautoimmune disease comprising isolating from a subject in need of suchtreatment CD8+ T cells that bind Hsp60_(p216) peptide; growing theisolated cells in a culture medium containing complexes of IL-15 andIL-15 receptor (IL-15C) until the number of CD8+ T cells that bindHsp60p216 peptide increases to at least 3-5% of CD8+ T cells, therebyproducing a population of cells enriched with CD8+ T cells that bindHsp60p216 peptide; and administering CD8+ T cells that bind Hsp60p₂₁₆peptide from the population of cells to the subject in an amounteffective to ameliorate a symptom of the autoimmune disease.

Expansion of CD8+ Treg cells specific for Qa-1(HLA-E)-Qdm orQa-1(HLA-E)-Hsp60_(p216) in vivo can be induced using a moietycomprising at least 4 units of Qa-1(HLA-E)-peptide complex. Accordingly,in some embodiments, the isolating step comprises sorting a samplecontaining T cells obtained from the subject into CD8+ T cells that bindHsp60_(p216) peptide using a fluorescently labeled moiety having atleast 4 units of a complex, wherein the complex is a majorhistocompatibility complex (MHC) class I antigen E (HLA-E) linked toHsp60_(p216) peptide. In some embodiments, the HLA-E-peptide complexcomprises biotin, and at least 4 such complexes associate throughbiotin-avidin interaction to form the moiety. In some embodiments, theavidin is fluorescently labeled. In some embodiments, the avidin islabeled with Phycoerythrin (PE). In some embodiments, the population ofcells enriched with CD8+ T cells that bind Hsp60p216 peptide is furtherenriched using anti-R-Phycoerythrin (PE) microbeads. Specifically, CD8 Tcells are first labeled with Qa-1-Hsp60p216 tetramers conjugated withPhycoerythrin (PE). These tetramer positive CD8 cells can then beselected by subsequent labeling with anti-PE antibody conjugated withmicrobeads and then by mounting them onto the magnetic field. Cells arethen collected by detaching from the magnetic field. This procedureenables the enrichment of Tet+ CD8 T cells from the CD8+ T cell pool.

A subject in need of treatment of autoimmune disease is a subjectidentified as having an autoimmune disease, i.e. the subject has beendiagnosed by a physician (e.g., using methods well known in the art) ashaving an autoimmune disease. In some embodiments, the subject in needof treatment is a subject suspected of having or developing anautoimmune disease, such as a subject presenting one or more symptomsindicative of an autoimmune disease. In some embodiments, a subjectsuspected of having an autoimmune disease may display abnormal titres ofautoantibodies. The subject having abnormal titres of autoantibodies mayhave at least one other symptom of autoimmune disease or may be withoutother symptoms associated with autoimmune disease. The term “subject inneed of treatment” further includes people who once had an autoimmunedisease but whose symptoms have ameliorated.

The subject is an animal, typically a mammal. In one aspect, the subjectis a dog, a cat, a horse, a sheep, a goat, a cow or a rodent. Inimportant embodiments, the subject is a human.

A self-antigen (or auto-antigen) is a subject's self-producedconstituent, against which the subject mounts an undesired immuneresponse. An “autoantibody” is an antibody produced by a subject, whichbinds to one or more of the subject's own constituents or self-antigens.The term ‘autoimmune disease’ refers to those disease states andconditions wherein the immune response of the patient is directedagainst the patient's own constituents resulting in an undesirable andoften terribly debilitating condition. As used herein, ‘autoimmunedisease’ is intended to further include autoimmune conditions, syndromesand the like. Example of autoimmune diseases include, but are notlimited to systemic lupus erythematosus, chronic graft versus hostdisease, rheumatoid arthritis, insulin-dependent diabetes mellitus,multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren'ssyndrome, Graves disease, Crohn's disease, Waldenstrom'smacroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy ofundetermined origin, POEMS syndrome, myeloma, macroglobulinemia, andcold agglutinin disease. In some embodiments, the autoimmune diseaseinvolves antibodies to a self-antigen and the subject has the antibodiesto the self-antigen.

The nanoparticles, Hsp60p216-loaded dendritic cells and the enrichedCD8+ T cells that bind Hsp60p216 peptide described herein can also beused to treat human immunodeficiency virus (HIV) infection. CD4+follicular helper T cells (TFH) are known to serve as the major CD4 Tcell compartment for HIV-1 infection, replication, and production (J ExpMed. 2013 Jan. 14; 210(1):143-56). As described herein, the specializedregulatory CD8+ T cells selectively suppress CD4+ follicular helper Tcell (TFH) activity through perforin-dependent lysis and elimination byrecognition of class Ib major histocompatibility complex (MHC) peptideQa-1 (mouse homolog of human leukocyte antigen E (HLA-E)) expressed atthe surface of TFH cells. It is believed that the CD8+ Treg cellsspecific for Qa-1(HLA-E)-Hsp60_(p216) target activated, HIV-infected TFHcells which present Hsp60p₂₁₆ on their surface. Thus, CD8+ cellsspecific for Hsp60p₂₁₆ peptide can be used to eliminate the TFH cellsthat serve as major reservoirs for HIV-1 infection. It is believed thatthe CD8+ Treg cells specific for Qa-1(HLA-E)-Hsp60_(p216) do notgenerally suppress the immune response, and instead, target activated,HIV-infected CD4+ follicular helper T cells (TFH) that present theHsp60_(p216) peptide on their surface. Accordingly, aspects of theinvention involve methods of treating HIV infection relate to thedevelopment of strategies based on in vivo and in vitro expansion andactivation of the CD8+ Treg cells for the treatment of HIV infection.

A subject in need of treatment of HIV infection is a subject identifiedas having HIV infection, i.e. the subject has been diagnosed by aphysician (e.g., using methods well known in the art) as having HIVinfection. In some embodiments, the subject in need of treatment is asubject suspected of having or developing HIV infection, such asinjection drug users who share needles, infants born to mothers with HIVinfection who did not receive HIV therapy during pregnancy, people whoreceived blood transfusions or clotting products between 1977 and 1985(before screening for the virus became standard practice), people whohave unprotected sex, especially with people who have other high-riskbehaviors, are HIV-positive, or have AIDS. The term “subject in need oftreatment” further includes people who were previously diagnosed withHIV infection and have started to present one or more symptoms of HIVinfection.

The subject is an animal, typically a mammal. In one aspect, the subjectis a dog, a cat, a horse, a sheep, a goat, a cow or a rodent. Inimportant embodiments, the subject is a human.

The nanoparticles, Hsp60p216-loaded dendritic cells and the enrichedCD8+ T cells that bind Hsp60p216 peptide described herein areadministered in an effective amount to treat autoimmune diseases or HIVinfection. An effective amount is a dose sufficient to provide amedically desirable result and can be determined by one of skill in theart using routine methods. In some embodiments, an effective amount isan amount which results in any improvement in the condition beingtreated. In some embodiments, an effective amount may depend on the typeand extent of the autoimmune disease or condition being treated and/oruse of one or more additional therapeutic agents. However, one of skillin the art can determine appropriate doses and ranges of therapeuticagents to use, for example based on in vitro and/or in vivo testingand/or other knowledge of compound dosages.

When administered to a subject, effective amounts of the therapeuticagent will depend, of course, on the particular disease being treated;the severity of the disease; individual patient parameters includingage, physical condition, size and weight, concurrent treatment,frequency of treatment, and the mode of administration. These factorsare well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. In someembodiments, a maximum dose is used, that is, the highest safe doseaccording to sound medical judgment.

In the treatment of autoimmune disease, an effective amount is thatamount which slows the progression of the disease, halts the progressionof the disease, or reverses the progression of the disease. An effectiveamount includes that amount necessary to slow, reduce, inhibit,ameliorate or reverse one or more symptoms associated with theautoimmune disease. In some embodiments, such terms refer to a reductionin the swelling of one or more joints or a reduction in the pain,fatigue and/or fever associated with an autoimmune disorder. In someembodiments, such terms refer to a reduction in the levels ofcirculating autoantibodies associated with the autoimmune disease. Insome embodiments, such terms refer to a reduction in a human's PASIscore. In some embodiments, such terms refer to an improvement in ahuman's global assessment score.

In the treatment of HIV infection, an effective amount is that amountwhich slows the progression of the disease, halts the progression of thedisease, or reverses the progression of the infection. An effectiveamount includes that amount necessary to slow, reduce, inhibit,ameliorate or reverse one or more symptoms associated with the HIVinfection.

An effective amount of a compound typically will vary from about 0.001mg/kg to about 1000 mg/kg in one or more dose administrations, for oneor several days (depending of course of the mode of administration andthe factors discussed above).

Actual dosage levels of the therapeutic agent can be varied to obtain anamount that is effective to achieve the desired therapeutic response fora particular patient, compositions, and mode of administration. Theselected dosage level depends upon the activity of the particularcompound, the route of administration, the tissue being treated, andprior medical history of the patient being treated. However, it iswithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effort and togradually increase the dosage until the desired effect is achieved.

Pharmaceutical preparations and compounds are administered to a subjectby any suitable route. For example, compositions can be administeredorally, including sublingually, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically andtransdermally (as by powders, ointments, or drops), bucally, or nasally.The pharmaceutical preparations of the present invention may include orbe diluted into a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible fillers, diluants or other such substances, which aresuitable for administration to a human or other mammal such as a dog,cat, or horse. The term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The carriers are capable ofbeing commingled with the preparations of the present invention, andwith each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficacy orstability. Carriers suitable for oral, subcutaneous, intravenous,intramuscular, etc. formulations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLE

A small subset of CD8 cells is essential for the maintenance ofself-tolerance and plays an important role in the inhibition ofautoimmune disease 1. This subset of regulatory T cells recognizesQa-1-peptide complexes that are upregulated by pathogenic CD4 T cells,resulting in elimination of these cells through perforin-dependentlysis. Development of strategies based on in vivo expansion andactivation of CD8+ Treg represents a new avenue of immunotherapy for thetreatment of autoimmune diseases.

Antigen Presentation by Qa-1 (HLA-E):

Two major peptides presented by Qa-1 (HLA-E) are Qdm (B7sp in human) andHsp60p216. Qdm (B7sp) is a peptide derived from the leader sequence ofMHC class Ia which can bind to both the TCR and to CD94/NKG2A receptors.The Hsp60_(p216) peptide can efficiently replace Qdm peptides underconditions of cellular activation and stress. A third peptide that ispresented by Qa-1 under conditions of defective antigen processing inthe endoplasmic reticulum (ERAAP dysfunction) is the highly conservedpeptide FL9. Recognition of Qa-1/FL9 by CD8 T cells may serve toeliminate abnormal cells that have defects in antigen presentation. Insum, Qa-1 dependent recognition represents a potential screening systemfor cells that are stressed or activated, as demonstrated in cases ofinfection and secondary to cellular defects of peptide processingassociated with autoimmunity and cellular transformation.

Qa-1-peptide tetramers have been generated to detect peptides-specificCD8+ T cells. In the case of Qa-1-Qdm tetramers, a Qa-1 proteincontaining a point mutation (R72A) was used to prevent binding ofQa-1-Qdm to CD94/NKG2A receptors and allow specific detection of CD8+ Tcells that express Qa-1 restricted TCR.

DC/Peptide Immunization and Inhibition of Autoimmune Disease:

Expansion of CD8 T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216) invivo can be induced by vaccination of mice with dendritic cells pulsedwith either peptide. WT B6 mice that were immunized with chickencollagen type II were treated by subcutaneous injection of irradiatedB6. Kb^(−/−)Db^(−/−)DC pulsed with either Hsp60_(p216) or Qdm peptidesat days 27, 36, and 49 and the progression of arthritis was monitored.Immunization with Hsp60_(p216)-loaded DC efficiently inhibits thedevelopment of collagen-induced arthritis (CIA), while vaccination withDC pulsed with Qdm peptide does not suppress disease progression (FIG.1A). Immunization of mice with Hsp60_(p216)-loaded Kb^(−/−)Db^(−/−)DCwas associated with expansion of Qa-1-Hsp60_(p216)-specific CD8+ T regcells as detected by Qa-1-Hsp60_(p216) tetramers (FIG. 1B). Althoughimmunization with Qdm-loaded Kb^(−/−)Db^(−/−)DC resulted in increasednumbers of Qa-1 R72A-Qdm tetramer+ cells (FIG. 1B), this immunizationdid not inhibit disease (FIG. 1A).

These findings indicate that specific in vivo expansion ofHsp60p216-specific CD8 Treg can contribute to the suppression ofpathogenic CD4 cells and inhibition of disease progression, a findingsupported by adoptive transfer studies of purified tetramer+Hsp60_(p216)-specific CD8+ T cells as described below.

Inhibition of Autoimmune Disease by Qa-1-Hsp60p216 Tetramer+ CD8 Cells:

To test the ability of Hsp60_(p216)-Qa-1-restricted CD8 cells to carryCD8+ T reg activity, Qa-1-Hsp60_(p216)-tetramer+ CD8 T cells wereseparated from the tetramer-fraction by consecutive FACS sorting andmicrobead selection (FIG. 2A). Adoptive transfer of enrichedQa-1-Hsp60_(p216)-tetramer+ CD8 cells (3.5×10⁴ cells), but notQa-1-Hsp60_(p216)-tetramer− CD8 cells into Rag2−/−Prf1−/− hosts alongwith CII-immune CD4 and B cells inhibited autoantibody production andhalted arthritis progression (FIG. 2B-D). Analysis of CD8 cells in theseadoptive hosts revealed an expanded population ofQa-1-Hsp60_(p216)-tetramer+ CD8 cells after transfer of tetramer+ butnot after transfer of tetramer− CD8 cells (FIG. 2E). In contrast,transfer of R72A Qa-1-Qdm tetramer+ CD8+ cells did not prevent diseaseprogression, consistent with the failure of Qdm-DC immunization toinhibit disease progression.

Effects of p-HLA-E-NP Vaccination in Humanized Mice:

Expansion of HLA-E/peptide specific CD8 cells by p-HLA-E-NP vaccinationis tested in NOD.Cg-B2m^(tm1Unc) Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NSB)mice expressing the HLA-E transgene (NSB-HLA-E) covalently linked toh132m using HLA-E/peptide tetramers. Humanized NSB-HLA-E mice that arereconstituted with human immune cells and vaccinated with p-HLA-E-NP aretested for the frequency of peptide specific human CD8 cells and theirsurface phenotype (CD45RO and KIR). Monitoring of the expansion ofHLA-E/HSP_(p216) specific CD8 T cells by detecting these cells usingtetramers in humanized mice can be extended to detection of Tetramer⁺CD8 cells in PBMC of patients.

Superior Feature of p-Qa-1 (HLA-E)-NP Vaccination:

Both Qa-1 in mice and HLA-E in men exhibit a limited polymorphismdistinct from MHC class Ia molecules that are highly polymorphic. Thehuman HLA-E gene, for example, is expressed one out of two alleles whichdiffer at a single amino acid. Therefore, a generalized immunotherapyusing p-Qa-1 (HLA-E)-NP-based vaccination for the treatment ofautoimmune disease represents a promising therapeutic strategy that doesnot depend on extensive individualized pMHC-NP design.

Qa-1 R72A-Qdm and Qa-1-Hsp60_(p216) tetramers were designed to detectCD8⁺ T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216). The tetramerswere used to enrich CD8 cells specific for these ligands and the cellswere tested for regulatory activity in the context of CIA. Vaccinationof mice with dendritic cells (DC) pulsed with defined peptides, resultedin expansion of CD8⁺ T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216)in vivo. Although immunization with Hsp60_(p216)-loaded DC efficientlyinhibited the development of CIA, vaccination with DC pulsed with Qdmpeptide did not suppress disease progression. Moreover, transfer of asmall number of Qa-1-Hsp60p216 tetramer⁺ CD8⁺ cells, but not R72AQa-1-Qdm tetramer⁺ CD8⁺ cells, inhibited disease progression. Alteredprocessing of MHC class Ia leader peptide in activated cells or stressedcells results in increased processing and presentation of alternatepeptides, including those derived from Hsp60, to Qa-1-restricted CD8⁺ Tcells. Inhibitory signaling associated with the interaction between theCD94/NKG2A receptor and the Qa-1-Qdm complex overrides TCR-dependentactivation by Qa-1-Qdm ligands.

In sum, amelioration of autoimmune arthritis may be achieved bytargeting arthrogenic T_(FH) and T_(H)17 cells through mobilization ofCD8⁺ Treg. CD8⁺ Treg infusion may be used for patients who developresistance to MTX treatment or relapse after MTX withdrawal. Moreover,the IL-15 dependence of CD8⁺ Treg may be exploited for development ofpersonalized CD8⁺ Treg-based cellular therapy to arthritis after invitro expansion, or direct peptide-based expansion of CD8⁺ Treg in vivo.The development of strategies based on specific expansion and activationof CD8⁺ Treg represents a new and potentially effective approach to thetreatment of autoimmune disease.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in the abovedescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing”,“involving”, and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

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We claim:
 1. A nanoparticle comprising a biocompatible polymer and acomplex, wherein the complex is a major histocompatibility complex (MHC)class I antigen E (HLA-E) linked to a peptide, and wherein theHLA-E-peptide complex is linked to the surface of the nanoparticle, andwherein the peptide is Hsp60p216 or FL9.
 2. The nanoparticle of claim 1,wherein at least 4 units, at least 5, at least 6, at least 7, at least8, at least 9 or at least 10 units of the HLA-E-peptide complex arelinked together to form a moiety and the moiety is linked to the surfaceof the nanoparticle.
 3. The nanoparticle of claim 1, wherein the peptideis linked to the HLA-E via a flexible linker.
 4. The nanoparticle ofclaim 1, wherein heavy chain and light chain (β-2 microglobulin) of theHLA-E are linked via a flexible linker.
 5. The nanoparticle of claim 1,wherein a low releasing dose of IL-15 is incorporated into thenanoparticle.